PROJECT SUMMARY Tuberculosis (TB, Mtb) remains the leading infectious disease killer globally, despite the availability of curative treatment for most individuals. Currently, individual care and TB control efforts are dichotomized around ?latent TB infection? and ?active TB disease? based on the absence or presence of clinical signs/symptoms and Mtb culture positivity. However, none of the available methods for identifying latent tuberculosis infection distinguishes the few individuals who will develop active, transmissible TB from the vast majority of individuals who never will become ill from TB or transmit to others. The ability to identify incipient TB ? TB which has progressed beyond bacterial containment associated with latency but not yet reached the burden of culture-positive, symptomatic active disease ? could have far-reaching impact in thwarting transmission and decreasing individual morbidity and mortality. The global scale-up of ultra-sensitive Mtb DNA detection assays, such as the recently WHO-recommended GeneXpert MTB/RIF Ultra (Ultra), presents a critical opportunity to identify this incipient, pre-cultivatable disease stage. In a recent diagnostic accuracy study, we found that Ultra diagnosed more patients with TB, but also detected Mtb DNA in sputum among 5% of symptomatic, culture- negative persons. We hypothesize that sputum Mtb DNA-positive/culture-negative individuals are more likely to have incipient TB than Mtb DNA-negative/culture-negative (TB-negative) individuals. To test this hypothesis, we propose a substudy to an R01- funded Ultra diagnostic accuracy study of adults in Africa with signs/symptoms of TB to evaluate the biology and natural history Ultra Mtb DNA-positive/culture-negative individuals. In the first two Aims, we will evaluate and compare biomarker correlates of TB disease risk between MTB DNA positive/culture negative and TB-negative individuals. In Aim 1, we will characterize host clinical and demographic factors associated with sputum Mtb DNA-positive/culture-negative status and characterize these individuals on the spectrum of Mtb infection using a pre-validated blood transcriptional signature predictive of progression to active TB. In Aim 2, we will evaluate and compare microbiologic indicators of TB disease between Mtb DNA positive/culture negative and TB-negative individuals. Specifically, we propose to investigate the presence of Mtb mRNA from sputum as a sentinel for transcriptionally-active Mtb populations among the symptomatic, untreated study population, and evaluate for revivable Mtb populations through culture supplementation of growth promoting factors from conditioned media. In addition to evaluation of biomarkers at study entry, we will evaluate the natural history of Mtb DNA- positive/culture-negative individuals with respect to TB-negative controls using longitudinal clinical, microbiologic, and host transcriptional data collected over a 12-month follow-up period in the parent study. Together, these proposed studies will inform the utility of ultrasensitive Mtb DNA detection tests for early TB risk stratification and guide implementation and interpretation of these tests as they emerge globally into the frontlines of TB diagnostic algorithms.